The present invention relates to a nucleic acid sequence encoding a chimeric hydroxyphenylpyruvate dioxygenase (HPPD), a chimeric gene comprising this sequence as encoding sequence, and its use for obtaining plants which are resistant to certain herbicides.
The hydroxyphenylpyruvate dioxegenases are enzymes which catalyse the transformation reaction of parahydroxyphenylpyruvate (HIPP) into homogentisate. This reaction takes place in the presence of iron (FE2+) and in the presence of oxygen (Crouch N.P. et al., Tetrahedron, 53, 20, 6993-7010, 1997). We may put forward the hypothesis that the HPPDs contain an active site which is suitable of catalyzing this reaction, in which the iron, the substrate and the water molecule combine, even though such an active site has never been described to date.
Moreover, there are also known certain molecules which inhibit this enzyme and which attach themselves competitively to the enzyme to inhibit the transformation of HPP into homogentisate. It has been found that some of these molecules can be used as herbicides, insofar as the inhibition of the reaction in the plants leads to bleaching of the leaves of the treated plants and to the death of these plants (Pallett K. E. et al., 1997, Pestic. Sci. 50 83-84). Such herbicides of the prior art which target HPPD are, in particular, the isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, which is a selective maize herbicide, the diketonitriles (EP 496 630, EP 496 631) in particular 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3-phenyl)-propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3-Cl2-phenyl)-propane-1,3-dione, the triketones (EP 625 505, EP 625 508, U.S. Pat. No. 5,506,195), in particular sulcotrione, or else the pyrazolinates.,
To make the plants herbicide-tolerant, three principal strategies are available, viz. (1) making the herbicide non-toxic using an enzyme which transforms the herbicide or its active metabolite into non-toxic degradation products, such as, for example, the enzymes for tolerance to bromoxynil or to Basta (EP 242 236, EP 337 899); (2) conversion of the target enzyme into a functional enzyme which is less sensitive to the herbicide or its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP 293 356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpression of the sensitive enzyme, in such a way that the plant produces high enough quantities of the target enzyme with regard to the kinetic constants of this enzyme relative to the herbicide in such a way that it has enough functional enzyme despite the presence of its inhibitor.
With this third strategy, it has been described that plants which are tolerant to HPPD inhibitors (WO 96/38567) were successfully obtained, it being understood that a simple strategy of overexpressing the sensitive (unaltered) target enzyme was successfully employed for the first time to impart to the plants a herbicide tolerance which is at an agronomical level.
Despite the success obtained with this simple strategy of overexpressing the target enzyme, the system for HPPD inhibitor tolerance must be varied to obtain tolerance whatever the culture conditions of the tolerant plants or the commercial doses of herbicide application in the field. It is known from the prior art (WO 96/38567) that enzymes of different origin (plants, bacteria, fungi) have primary protein sequences which differ substantially and that these enzymes have an identical function and essentially similar or related kinetic characteristics.
It has now been found that all these HPPDs have, on the one hand, many sequence homologies in their C-terminal portion (FIG. 1) and, on the other hand, an essentially similar tertiary (three-dimensional) structure (FIG. 2). As regards the competitive inhibition characteristic, the hypothesis is put forward that the HPPD inhibitors attach themselves to the enzyme at the active site of the latter, or in its vicinity, in such a way that the access of HPP to this active site is blocked and its conversion in the presence of iron and of water is prevented. It has now been found that, by mutating the enzyme in its C-terminal portion, it was possible to obtain functional HPPDs which are less sensitive to HPPD inhibitors, so that their expression in the plants allows an improved HPPD inhibitor tolerance. As regards these elements, it can thus be concluded that the active site of the enzyme is located in its C-terminal portion, while its N-terminal portion essentially ensures its stability and its oligomerization (Pseudomonas HPPD is a tetramer, plant HPPDs are dimers).
It has now been found that it was possible to generate a chimeric enzyme by combining the N-tenninal portion of a first enzyme with the C-terminal position of a second enzyme so as to obtain a novel functional chimeric HPPD, which allows each portion to be selected for its particular properties, such as, for example, to select the N-terminal portion of a first enzyme for its stability properties in a given cell (plant, bacterium and the like) and the C-terminal portion of a second enzyme for its kinetic properties (activity, inhibitor tolerance, and the like).
The present invention therefore primarily relates to a chimeric HPPD which, while being functional, that is to say retaining its properties of catalysing the conversion of HPP into homogentisate, comprises the N-terminal portion of a first HPPD in combination with the C-terminal portion of a second HPPD.